The use of microorganisms, particularly E. coli, to express foreign genes for protein production has been commonplace for many years. Since, in most cases, the purpose for using the microorganisms is to permit the production of large quantities of the protein for commercial purposes, it is usually desired to express the protein at the highest possible level. For this reason, most expression systems and the DNA constructs used therein, are specifically adapted for high level expression. However, there are situations in which low to moderate level expression is actually more desirable, or even essential, For example, overexpression of some genes is lethal. Also, in cases in which microorganisms are used as the basis for a screen to detect action of a drug against a particular gene product, a low level of expression of the protein provides for enhanced sensitivity. However, fine-tuning the level of expression is not a routine task.
As an example, it is desirable to achieve low to moderate levels of expression of the genes encoding tetracycline resistance in order to develop appropriate microorganisms for screening drugs that overcome tetracycline resistance. This resistance in the majority of microorganisms is the result of an energy-dependent efflux system (1). These efflux pumps have been analyzed in a variety of both Gram-negative and Gram-positive bacteria, and all have shown a similar secondary structure with multiple membrane spanning domains. Nonetheless comparison of the amino acid sequence of the most common Gram-negative pump, as encoded by tetA gene of E. coli from transposon Tn10, and the tetK gene from Gram-positive Staphylococcus aureus shows little identity (2). However, since these two pumps perform similar functions, it would be useful to perform studies on the tetK gene encoding the tetracycline efflux pump of S. aureus in an E. coli host, given the ease of performing genetic manipulations and biochemical studies in this system. The tetA and tetK efflux pumps are responsible for most of the clinical problems associated with tetracycline efflux. In addition, the use of isogenic strains allows better comparison of the two efflux pumps.
A problem exists, however, if the tetA or tetK genes are cloned into a standard strong expression vector. Overexpression of the tetA gene from transposon Tn10 is lethal to E. coli, e.g., if this gene is expressed in a multicopy plasmid (3, 4). In Gram-negative bacteria, regulation of the pump is mediated by the tetR gene product, a repressor, located adjacent to a common regulatory region for tetA and tetR (5, 6). Therefore, assuming expression of other genes encoding efflux pumps, such as tetK, could be achieved, it is possible that full expression would also be lethal to E. coli.
Attempts to modify the Tn10 system to permit controlled expression of the tetA from transposon Tn10gene have been made. Eckert and Beck (7) have recently cloned and expressed tetA from transposon TN10 on a multicopy plasmid in the absence of tetR, using a regulated inducible expression system. In this system when tetA is fully induced, the cells again die, perhaps due to the dissipation of the proton motive force (7); active efflux of tetracycline out of bacteria is energized by the entry of a proton into the cell, but full induction apparently leads to the loss of the proton gradient essential to the bacteria's survival. In the Eckert and Beck system, the tetA gene is regulated at the level of transcription using a regulatory region containing the strong tac promoter and the lacI gene (lactose repressor) and the lac operator site on the multicopy plasmid pCB258. Expression of the tetA gene can be regulated using different concentrations of isopropyl-B-D-thiogalactopyranoside (IPTG).
Unfortunately, the Eckert and Beck system is unsuitable for the purpose of building an optimal screening organism for detection of inhibitors of the tetracycline efflux pump. First, restriction analysis of the plasmid pCB258 indicates that one of the two tetR operator sites of the tet regulatory region remain in the plasmid adjacent to the tetA coding region. Thus, the tetA gene is regulated both by the lac repressor as well as the tetracycline repressor, if both repressors are present in the cell. The presence of both repressors causes deleterious consequences in an expression system designed for use as a screening organism. Thus, although pCB258 does permit relatively weakened expression of the tetA gene, the level of tetracycline resistance is nonetheless too high for use in screening for pump inhibitors. Moreover, there is no convenient restriction site in the appropriate region to permit insertion of an alternate gene such as tetK.
A second problem arises, specifically with respect to expression of the S. aureus tetK gene in E. coli. The tetK gene has previously been cloned into an E. coli system and did not confer resistance (8). Thus, evidence indicates that the tetK gene cannot be expressed utilizing the natural S. aureus expression signals, such as the native TTG start codon. Therefore, some manipulation of the tetK gene is necessary to achieve expression in E. coli.
In order to overcome these difficulties, the present invention provides DNA constructs, vectors and E. coli host cells which result in low to moderate levels of proteins encoded therein, particularly heterologous proteins. Such materials are particularly useful in creation of screening assays for inhibitions of tetracycline resistance.